Chromatography devices and methods

Abstract

A device for automated or manual separations of complex chemical or biological mixtures and methods of use are described. The device is a chromatography pipette tip or column having a size exclusion media therein and held in place by a hydrophilic filter at the distal end of the pipette tip and an optional removable or pierceable barrier at the proximal end of the pipette tip. The size exclusion media is wetted by simply placing the tip into solvent and allowing capillary action to flow through the filter and into the SEC resin. The solvent is then allowed to gravity drain from the pipette tip, allowing the size exclusion media to reproducibly pack into a homogenous gel. Separations can then be performed with the gelled size exclusion media.

1 . A method of preparing a chromatography column, comprising:
 a) inserting a chromatography column into a container of polar fluid, wherein said chromatography column contains a hydrophilic filter and a dry chromatography resin above said hydrophilic filter, wherein said hydrophilic filter is positioned at a distal end of said chromatography column so as to have <50 μl dead volume below said hydrophilic filter;   b) wetting said hydrophilic filter by capillary action pulling said polar fluid up through said hydrophilic filter and into said dry chromatography resin to form swelled resin;   c) equilibrating said swelled resin in said polar fluid for a time sufficient to form a homogenous wetted resin (gel);   d) optionally adding a small volume of said polar fluid to a top end of said chromatography column; and   e) draining any excess polar fluid out of said distal end of said chromatography column.   
     
     
         2 . The method of  claim 1 , wherein said chromatography column is a pipette tip. 
     
     
         3 . The method of  claim 1 , wherein step d) is performed on a robotic liquid handling platform by a robotic liquid handler. 
     
     
         4 . The method of  claim 1 , wherein said polar fluid is an aqueous buffer having a pH of 3-12 or said polar fluid is phosphate buffered saline. 
     
     
         5 . The method of  claim 1 , wherein said hydrophilic filter is a porous plastic frit that has been modified with a surfactant, polysorbate, polyethylene glycol or polyvinyl alcohol or combinations thereof. 
     
     
         6 . The method of  claim 1 , wherein said hydrophilic filter is a mesh screen, porous polymeric material, porous glass, porous membrane, glass microfibers, glass capillaries, or porous ceramic. 
     
     
         7 . The method of  claim 1 , wherein said dry chromatography resin comprises agarose, sepharose, polyacrylamide, dextran, dextran crosslinked with epichlorohydrin, polystyrene, polyacrylate, cellulose, or a combination thereof. 
     
     
         8 . The method of  claim 1 , wherein said draining step comprises gravity flow or by applying positive pressure to said top end of said chromatography column. 
     
     
         9 . A method of chromatographic analyte separation, comprising:
 a) inserting a chromatography pipette tip into a container of polar fluid, wherein said chromatography pipette tip is a pipette tip containing a hydrophilic filter and a dry chromatography resin above said hydrophilic filter, wherein said hydrophilic filter is positioned at a distal end of said pipette tip so as to have <10 μl dead volume below said hydrophilic filter;   b) wetting said hydrophilic filter by capillary action pulling said polar fluid up through said hydrophilic filter and into said dry chromatography resin to form a swelled resin;   c) equilibrating said swelled resin in said polar fluid for a time sufficient to form a homogenous wet resin (gel);   d) optionally adding a small volume of said polar fluid to a top end of said chromatography pipette tip;   e) draining any excess polar fluid out of said distal end of said chromatography pipette tip;   f) adding a sample solution in said polar fluid to the top end of said chromatography pipette tip, said sample solution containing two or more analytes;   g) allowing said sample solution to load into said gel by gravity flow or by applying positive pressure;   h) optionally adding a wash solution (void volume) to the top end of said chromatography pipette tip, and allowing said wash solution to flow via gravity or positive pressure;   i) adding an elution solution (collection fraction) to the top end of said chromatography pipette tip; and   j) collecting one or more elution fractions having separated analytes via gravity flow or positive pressure in one or more containers.   
     
     
         10 . The method of  claim 9 , further comprising the step of analyzing said separated analytes using enzymatic assay, immunoassay, affinity assay, refractive index detector, UV-Vis detector, viscometer, multi-angle light scattering detector, gel electrophoresis, mass spectrometer and combination(s) thereof. 
     
     
         11 . The method of  claim 9 , wherein a pipetting aid is used in adding steps d), f), h) and i) and wherein said pipetting aid is a hand-held pipettor, a syringe, or a robotic liquid handler that directly attaches to a top end of said chromatography pipette tip, or indirectly attaches thereto through the use of an adaptor or a top-pipette-tip. 
     
     
         12 . The method of  claim 11 , wherein said pipetting aid is a robotic liquid handler and steps d) through j) are performed on a robotic liquid handling platform. 
     
     
         13 . The method of  claim 9 , wherein said sample solution, said wash solution and said elution solution are buffered to a pH of 3-12. 
     
     
         14 . The method of  claim 9 , wherein adding said elution solution step i) or adding said wash solution step h) or both are repeated 2 to 5 times. 
     
     
         15 . The method of  claim 9 , wherein said hydrophilic filter is selected from a screen, porous frit, porous polymeric material, porous membrane, porous glass, sintered glass, glass microfibers, glass capillaries, or porous ceramic. 
     
     
         16 . The method of  claim 9 , wherein said dry chromatography resin comprises agarose, sepharose, polyacrylamide, dextran, dextran crosslinked with epichlorohydrin, polystyrene, polyacrylate, cellulose, or a combination thereof. 
     
     
         17 . A chromatography pipette tip, comprising:
 a pipette tip comprising a hollow tube with an open top end over a conical section and a bottom end that is smaller than said top end;   said top end sized to fit over a separate pipette head;   an exterior surface of said pipette tip above said conical section having an annular ridge or three or more vertical fins;   a hydrophilic filter and a dry chromatography resin or media above said hydrophilic filter;   said hydrophilic filter positioned at said bottom end of said pipette tip so as to have less than 10 μl dead volume inside said pipette tip and below said hydrophilic filter; and   a barrier at said top end to retain said dry chromatography resin.   
     
     
         18 . The chromatography pipette tip of  claim 17 , wherein said hydrophilic filter is a porous plastic frit, a mesh screen, porous polymeric material, porous membrane, porous glass, glass microfibers, glass capillaries, or porous ceramic. 
     
     
         19 . The chromatography pipette tip of  claim 17 , wherein said dry chromatography resin is a dextran crosslinked with epichlorohydrin based size exclusion resin. 
     
     
         20 . A kit comprising a box with an array of holes therein, the chromatography pipette tip of  claim 17  reversibly inserted into each hole of said array of holes, plus instructions for use of said chromatography pipette tip.